This Project: This web page originated as an assignment in Emory University's Biology 142 lab course. Students were assigned proteins of interest and asked to research what is known about the protein and to examine whether the newly sequenced whale shark genome had evidence of an orthologous protein.
Background Information: THEM173/ the STING protein aka Trans-membrane protein 173 codes for a protein that is a facilitator of immune signaling and promotes the secretion of interferon type 1. There are some diseases associated with mutations associated with an alteration in this gene, especially given the fact that it plays a key role in detecting DNA in many viruses and bacteria by binding to cyclic di-GMP's , a kind of messenger produced by many bacteria.
Methods: The amino acid sequence of the gene was found using Ensembl databases by using the gene’s Ensemble ID (ENSP00000331288). This was done by entering the Ensemble ID, and then selecting ‘human’ as the species to search against. A transcript associated with the specific protein ID was chosen. BLASTp was then performed on the amino acid sequence. Once a good match was found from the BLAST results, similar sequences in other species were found using the NCBI Blast program (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Homologues were found in particular species by selecting the ‘protein blast’ option and running the search. The FASTA format of the sequence was inserted into the ‘query sequence’ box, ‘blastp’ was the selected tab, and the sequence was “BLASTed.” The whale shark database was imported from the Galaxy server (whaleshark.georgiaaquarium.org). Imported data appeared as a green numbered item. The human sequence of the gene was entered as the query sequence by selecting ‘get data’ and then ‘upload data.’ The FASTA format of the sequence was then entered into the text box and then executed. Once the whale shark database and the query sequence were both uploaded to the history, a BLAST search was performed by clicking ‘NCBI Blast+.’ The pasted sequence was chosen as the query and the ‘FASTA file from history’ was chosen as the subject database. E-value and length of alignment were used to determine if any of the results were significant hits in the whale shark database. Once a significant BLAST hit was discovered, its sequence was retrieved. ‘Whale-shark.predicted-genes’ database was selected and the sequence ID of the predicted gene was entered. To align sequences, Clustal algorithm was used. The sequence alignment was then used to create the phylogenetic tree.
Sequencing
Figure 1
photo Screen Shot 2015-04-12 at 6.47.37 PM_zpsctxjrkcp.png
Figure 1 illustrates the 6 matches that we found when cross-referencing the human TMEM gene with the whale shark sequence.
Domains:
Figure 2 Figure 2 shows the only single domain that was found, STING_C. Stimulator of interferon genes (STING) is a chief regulator that mediates cytokine production in response to microbial invasion. It performs this by directly sensing bacterial secondary messengers. It is also a signaling adaptor in the IFN response to cytosolic DNA. STING is found in the endoplasmic reticulum.
Protein Domain: The potential matches founded through blasting the TMEM 173/ STING protein have been shown to contain the TMEM 173 and STING_C domains. The TMEM 173 domain includes matches that are transmembrane adaptors which are involved in innate immune signaling process and induce type 1 interferons. The STING_C domain are described as regulators that mediates the cytokine (cells involved in signaling) production when during a microbial invasion occurs.
Orthologs:
Column1
Column2
Column3
Column4
Column5
Column6
Column7
Column8
Column9
Species
Description
Max Score
Total Score
Query coverage
E-Value
Identity
Accession
Zebra Fish
PREDICTED: stimulator of interferon genes protein isoform X1
182
182
85%
4.00E-52
36%
XP 005157178.1
Clawed Frog
Stimulator of interferon genes protein
248
248
88%
2.00E-78
41%
NP 001106445.2
Elephant Shark
PREDICTED: stimulator of interferon genes protein
236
236
97%
2.00E-73
39%
XP 007904451.1
Human
PREDICTED: stimulator of interferon genes protein isoform X3
530
530
68%
0.00E+00
100%
XP 011535942.1
Hits with the Whale Shark Genome We observed from the hits we received when we blasted the pasted sequence of the human STING gene to the whale shark sequences, the first column was the percent identity to the Query, the second column is the matching alignment of amino acids, and the most important column is the ninth column, which shows the e-value of the comparison. The closer the e-value is to zero, the less likely the overlap of amino acid sequences is due to just pure chance.
According to the hits we found from the galaxy site seen in the table above, there is no clear indication of any one gene in the whale shark that correlates well with the human STING gene, so the best thing to do at this point was to cross reference some of the hits that we found in the whale shark genome to other species and see if there are any matches to be found.
Phylogeny:
Figure 2 Figure 2 shows a phylogenetic tree showing the closest species to STING protein and its accordance to the whale shark. The length of each branch shows the relative similarity between each species
Conclusion: The TMEM173 codes for a protein that facilitates immune signaling, but is infact more common among apes and humans than in marine mammals. As a result we could not find the exact or similar STING gene inside the whale shark but instead decided to cross reference the TMEM173 gene with other species with its accordance to the whale shark. The phylogenetic tree accurately displays how distant the Human gene is from the Whale shark. Through this experiment, we were able to suggest that despite the absence of the STING protein inside the whale shark and the whale shark's closest relatives the Elephant shark contains it, that this gene had been developed as species evolved and deviated from the original branch from the evolutionary tree
6.) Ouyang, Songying, Xianqiang Song, and Yaya Wang. "Structural Analysis of the STING Adaptor Protein Reveals a Hydrophobic Dimer Interface and Mode of Cyclic Di-GMP Binding."Science Direct. Immunity, 29 June 2009. Web. 31 Mar. 2015.
7.) "Stimulator of Interferon Genes Protein."TMEM173. Uniprot, 4 Mar. 2015. Web. 31 Mar. 2015.
This Project:
This web page originated as an assignment in Emory University's Biology 142 lab course. Students were assigned proteins of interest and asked to research what is known about the protein and to examine whether the newly sequenced whale shark genome had evidence of an orthologous protein.
Background Information:
THEM173/ the STING protein aka Trans-membrane protein 173 codes for a protein that is a facilitator of immune signaling and promotes the secretion of interferon type 1. There are some diseases associated with mutations associated with an alteration in this gene, especially given the fact that it plays a key role in detecting DNA in many viruses and bacteria by binding to cyclic di-GMP's , a kind of messenger produced by many bacteria.
Methods:
The amino acid sequence of the gene was found using Ensembl databases by using the gene’s Ensemble ID (ENSP00000331288). This was done by entering the Ensemble ID, and then selecting ‘human’ as the species to search against. A transcript associated with the specific protein ID was chosen. BLASTp was then performed on the amino acid sequence. Once a good match was found from the BLAST results, similar sequences in other species were found using the NCBI Blast program (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Homologues were found in particular species by selecting the ‘protein blast’ option and running the search. The FASTA format of the sequence was inserted into the ‘query sequence’ box, ‘blastp’ was the selected tab, and the sequence was “BLASTed.” The whale shark database was imported from the Galaxy server (whaleshark.georgiaaquarium.org). Imported data appeared as a green numbered item. The human sequence of the gene was entered as the query sequence by selecting ‘get data’ and then ‘upload data.’ The FASTA format of the sequence was then entered into the text box and then executed. Once the whale shark database and the query sequence were both uploaded to the history, a BLAST search was performed by clicking ‘NCBI Blast+.’ The pasted sequence was chosen as the query and the ‘FASTA file from history’ was chosen as the subject database. E-value and length of alignment were used to determine if any of the results were significant hits in the whale shark database. Once a significant BLAST hit was discovered, its sequence was retrieved. ‘Whale-shark.predicted-genes’ database was selected and the sequence ID of the predicted gene was entered. To align sequences, Clustal algorithm was used. The sequence alignment was then used to create the phylogenetic tree.
Sequencing
Figure 1
Figure 1 illustrates the 6 matches that we found when cross-referencing the human TMEM gene with the whale shark sequence.
Domains:
Figure 2
Figure 2 shows the only single domain that was found, STING_C. Stimulator of interferon genes (STING) is a chief regulator that mediates cytokine production in response to microbial invasion. It performs this by directly sensing bacterial secondary messengers. It is also a signaling adaptor in the IFN response to cytosolic DNA. STING is found in the endoplasmic reticulum.
Protein Domain: The potential matches founded through blasting the TMEM 173/ STING protein have been shown to contain the TMEM 173 and STING_C domains. The TMEM 173 domain includes matches that are transmembrane adaptors which are involved in innate immune signaling process and induce type 1 interferons. The STING_C domain are described as regulators that mediates the cytokine (cells involved in signaling) production when during a microbial invasion occurs.
Orthologs:
Hits with the Whale Shark Genome
We observed from the hits we received when we blasted the pasted sequence of the human STING gene to the whale shark sequences, the first column was the percent identity to the Query, the second column is the matching alignment of amino acids, and the most important column is the ninth column, which shows the e-value of the comparison. The closer the e-value is to zero, the less likely the overlap of amino acid sequences is due to just pure
chance.
According to the hits we found from the galaxy site seen in the table above, there is no clear indication of any one gene in the whale shark that correlates well with the human STING gene, so the best thing to do at this point was to cross reference some of the hits that we found in the whale shark genome to other species and see if there are any matches to be found.
Phylogeny:
Figure 2
Figure 2 shows a phylogenetic tree showing the closest species to STING protein and its accordance to the whale shark. The length of each branch shows the relative similarity between each species
Conclusion: The TMEM173 codes for a protein that facilitates immune signaling, but is infact more common among apes and humans than in marine mammals. As a result we could not find the exact or similar STING gene inside the whale shark but instead decided to cross reference the TMEM173 gene with other species with its accordance to the whale shark. The phylogenetic tree accurately displays how distant the Human gene is from the Whale shark. Through this experiment, we were able to suggest that despite the absence of the STING protein inside the whale shark and the whale shark's closest relatives the Elephant shark contains it, that this gene had been developed as species evolved and deviated from the original branch from the evolutionary tree
Reference:
1.) "Basic Local Alignment Search Tool." BLAST:. N.p., n.d. Web. 14 Apr. 2015.
2.) "Ensembl Genome Browser." Ensembl Genome Browser. N.p., n.d. Web. 14 Apr. 2015.
3.)Galaxy/ Whale Shark. Coca Cola/ Georgia Aquarium, n.d. Web. 14 Apr. 2015. <http://whaleshark.georgiaaquarium.org/>.
4) "Genes and Mapped Phenotypes." National Center for Biotechnology Information. U.S. National Library of Medicine, 24 Mar. 2014. Web. 31 Mar. 2015.
5.) Multiple Sequence Alignment by CLUSTALW. CLUSTALW, n.d. Web. 14 Apr. 2015 <http://www.genome.jp/tools/clustalw/>.
6.) Ouyang, Songying, Xianqiang Song, and Yaya Wang. "Structural Analysis of the STING Adaptor Protein Reveals a Hydrophobic Dimer Interface and Mode of Cyclic Di-GMP Binding." Science Direct. Immunity, 29 June 2009. Web. 31 Mar. 2015.
7.) "Stimulator of Interferon Genes Protein." TMEM173. Uniprot, 4 Mar. 2015. Web. 31 Mar. 2015.